1. Field of the Invention
This invention relates to a radioimmunoassay method.
2. Discussion of the Background
Conventional "sandwich" immunoassays generally employ the reaction between an antigen and a first antibody immobilized on an insoluble solid, then a second antibody labeled with a radioisotope or enzyme is reacted with the immuno complex, and finally the radioactivity or enzymatic activity is measured. Among sandwich assays which use a second antibody labeled with an enzyme, there is no limit to the amount of labeled second antibody that can be used, so that even when the second antibody does not have high sensitivity (i.e., the dissociation constant between antibody and antigen is large) the overall sensitivity of the assay can be enhanced by using the second antibody in high concentration. However, enzyme activity is harder to detect than the specific activity of radioisotopes, so that extremely high sensitivity cannot generally be obtained with enzymatic systems. Thus, there has been employed an improved method in which many labeled enzymes are bound per one molecule of the "second antibody" used to bind to an antigen, e.g. the avidin biotin complex method (Practice and Theory of Enzyme Immunoassays, P. Tijssen, Elsevier, 1985). However, even when these methods are used, measurement sensitivity in enzyme immunoassays cannot equal the sensitivity of a radioactive label, and it is difficult to obtain a measurement system with high sensitivity (see Japanese Provisional Patent Publication No. 118656/1988).
On the other hand, radioimmunoassays using a second antibody labeled with a radioisotope provide high measurement sensitivity since radioactivity can be detected at a lower concentration as compared with enzymic activity. However, the amount of radioactivity that can be added to a measurement system is limited, so that the concentration of the second antibody is limited to relatively low values. Furthermore, when the only available second antibody has inherently low sensitivity, the second antibody does not bind sufficiently to the antigen, therefore the overall sensitivity of the measurement system cannot be improved sufficiently by merely raising the concentration of the second antibody.
To describe the situation more specifically, the ratio of antigen bound to a second antibody relative to the amount of antigen not bound to a second antibody ([AgAb2]/[Ag]) in a reaction mixture is determined by the concentration of the second antibody [Ab2] and a dissociation constant between the second antibody and the antigen (Kd) as shown in the following formula. ##EQU1##
That is, the higher the concentration of second antibody and the smaller the dissociation constant, the larger the amount of second antibody that is bound to the antigen, whereby measurement sensitivity is heightened.
However, the dose of radioactivity which can be used in a measurement system is limited, so that the concentration of the second antibody is limited. When the dissociation constant of the second antibody is large, a measurement system having sufficient sensitivity cannot be obtained (Kodo Obata et al., The Japanese Journal of "Nuclear Medicine", Vol. 26, No. 3 (1989)).
As described above, it has conventionally been considered very important to obtain a second antibody with high sensitivity in order to obtain an immunoassay system with high sensitivity. However, it is often difficult to obtain an antibody with sufficiently high sensitivity, and great efforts have been made therefor.
Further, in conventional techniques, the second antibody is labeled directly. Thus, when two or more measurement systems are to be prepared in order to measure two or more antigens, then two or more secondary antibodies which identify the respective antigens must be labeled separately. This requirement results in an increase in cost.